Study of the mechanisms involved in the acquired resistance to the monoclonal antibody isatuximab in multiple myeloma
Marta González Rodríguez
Centro de Investigación del Cáncer (CSIC-USAL-FICUS), Salamanca
Introduction: Multiple myeloma (MM) is a hematological malignancy characterized by the clonal proliferation of plasma cells in the bone marrow. In recent years, numerous immunotherapeutic treatments have been developed, most notably monoclonal antibodies (mAbs). However, the development of resistance to these drugs constitutes the main cause of relapse in patients.
Objectives: To study the mechanisms involved in resistance to two of the mechanisms of action of the anti-CD38 mAb isatuximab: the direct activity of the drug on MM cells and the complement-dependent cytotoxicity (CDC).
Methodology: A cellular model of resistance was generated through the prolonged exposure of the sensitive MOLP-8 cell line to increasing doses of isatuximab under two conditions: in the absence of human serum (RDA cell line, resistant to the direct activity), and in the presence of human serum as a source of complement (RCDC cell line, resistant to the CDC). Among the studies carried out for the characterization of these resistant cell lines, gene expression differences between the sensitive and resistant lines were assessed using microarrays (Clariom S Array, Human).
Results: First, we confirmed that the generated cell lines were resistant to both the direct activity and the CDC of isatuximab. Both cell lines also showed cross-resistance to CDC and apoptosis via crosslinking mediated by daratumumab, another anti-CD38 mAb used in MM treatment. We assessed that resistant cells presented a lower expression of CD38, the target of isatuximab, at both the mRNA and protein levels; however, enforced expression of CD38 failed to resensitize the resistant cells to isatuximab.
Gene expression analysis revealed an overexpression of the antiapoptotic protein BCL-2 in both resistant cell lines. In addition, the RDA cell line presented a notable overexpression of cyclin D2 (CCND2), whereas the RCDC line showed alterations in the mismatch repair, homologous recombination and cell cycle pathways
In relation with the mentioned deregulated gene expression profiles, we observed that the resistant cell lines had a higher proliferation rate associated to an increased number of cells in the S phase and a decrease in the G2/M phase. The increased expression of CCND2 in the RDA cell line correlated with the downregulation of miRNAs 15a and 16, which regulate CCND2 expression, and with the expression of a shorter CCND2 mRNA isoform lacking the 3´UTR miRNA binding/regulatory sites. In contrast, the RCDC cell line expressed higher levels of miRNAs 15a and 16 and predominance of the long CCND2 mRNA isoform, leading to expression of both CCND1 and CCND2 at similar levels to the parental MOLP-8 cell line. Nevertheless, this dependency on CCND1 and CCND2 of the resistant cell lines led to a relative sensitivity to treatment with the CDK4/6 inhibitor palbociclib.
To gain insight into alterations related to the DNA damage response, resistant cells were subjected to treatment with the alkylating agent melphalan. Both RDA and RCDC cell lines evaded the DNA damage checkpoint accumulating DNA damage, and preventing p53 phosphorylation and cell cycle arrest. Although this accumulation of DNA damage generated a pro-apoptotic intracellular environment with elevated levels of PUMA, resistant cell lines seem to overcome apoptosis due to the compensatory overexpression of the anti-apoptotic protein BCL-2. Despite this, the elevated expression of BCL-2 in RDA and RCDC cell lines represented a therapeutically exploitable vulnerability, since both cell models displayed marked sensitivity to the BCL-2 inhibitor venetoclax.
Conclusions: Cell lines resistant to the isatuximab-mediated direct activity and CDC present a decrease in CD38 expression at transcriptional and translational levels, concomitant with a deregulated gene expression profile leading to increased proliferation and/or DNA damage response evasion and the activation of anti-apoptotic pathways. Despite these resistance mechanisms, we found new vulnerabilities which may be pharmacologically addressed in these resistant cells.